Immunoassays have become methods of choice for rapid in vitro diagnostics due in part to the ease of use, speed of reaction, and relatively low cost. A common immunoassay format comprises a “capture” reagent immobilized on a solid substrate wherein the capture reagent is capable of binding the target analyte, or a complex containing the target analyte, or a molecule that competes with the target analyte for binding sites on the capture reagent. By localizing the capture reagent in a restricted zone or area, the result of the immunoassay is readily visualized or detected.
In particular, immunoassays in the form of lateral flow test strips for the rapid analysis of fluids, including, but not limited to, body fluids such as urine, saliva, serum, plasma, whole blood, spinal fluid, amniotic fluid, and the like, or liquid extracts of specimens such as feces, sputum, nasal swabs, etc., are popular as a means of determining the presence or absence of target analytes, such as hormones, drugs, allergens, and disease-related antigens. Examples of lateral flow devices are disclosed in U.S. Pat. Nos. 4,094,647, 4,235,601, and 4,361,537 to Deutsch et al., as well as U.S. Pat. No. 5,120,643 to Ching et al., U.S. Pat. No. 5,602,040 to May et al., U.S. Pat. No. 5,714,389 to Charlton et al., and U.S. Pat. No. 6,352,862 to Davis et al., among many others.
Regarding ease of use, typically very little, if any, specimen processing is required prior to adding a liquid sample to a test strip. Therefore, preferably, the sensitivity of a lateral flow test device is adequate to detect or measure a minimal biologically and/or physiologically significant concentration of particular target analyte, as may be expected to be contained within a fluid specimen as collected, without requiring additional processing to concentrate the target analyte prior to testing. Such restrictions on processing present a challenge when the biologically or physiologically meaningful concentration of a target analyte is low. Also, throat or nasal swabs, sputum, semen, vaginal and cervical secretions and other sorts of viscous specimens usually require addition of a reagent to extract, breakdown or “thin” the sample to achieve an appropriately liquid state to flow through a test device. Addition of such reagents leads to further dilution of the concentration of target analytes, compounding the problem of detecting low levels of target analytes that may be present in a mucus specimen, throat swab, semen, and the like.
Methods of increasing the amount of detectable label captured at a test site have been described. U.S. Pat. No. 5,141,850 to Cole et al., expressly incorporated by reference herein in its entirety, teaches the binding of a “capturable” reagent to the target analyte, along with binding of a labeled reagent, and the immobilization of a capture reagent specific for the capturable component. A preferred embodiment uses streptavidin conjugated to latex particles and localized within the pores of the porous carrier material at the test site by depositing the particles under mild vacuum conditions. However, conjugation of streptavidin to latex particles adds to the labor and cost of manufacturing the test device and entrapping particles within the pores of the carrier material is not equivalent to chemically or physically binding, directly or indirectly, the capture reagent to the carrier material. Moreover, entrapment of latex particles within a carrier material requires porosity sufficient to contain particles, but is not desirable for use in connection with samples containing cellular contaminants and/or debris, such as sputum samples, semen, vagina and cervical secretions, whole blood, and the like.
Another approach to amplification of detection within an immunoassay is to use polymeric carriers in preparing various conjugated reagents. Such polymeric conjugates are disclosed in U.S. Pat. No. 5,543,332 to Lihme et al. (expressly incorporated by reference herein in its entirety). One example is a dextran-polystreptavidin conjugate. Conjugation of multiple molecules of streptavidin to each dextran molecule significantly increases the number of available streptavidin molecules within a localized area for binding to a receptor molecule, namely biotin, thereby amplifying the amount of biotinylated molecules captured within a localized area.
It is known to use dextran-polystreptavidin for preparing conjugate reagents that react in a “free” form (not immobilized on a solid support) in solution, for binding to or competing with a target analyte. Such reagents are taught in U.S. Pat. No. 5,543,332 in connection with ELISA and “dot-blot” immunoassays wherein biotin is immobilized on a solid support and, thus, biotin serves as the capture reagent. More recently, polymeric conjugates are described in U.S. Pat. No. 6,709,611 to Lassen et al. in connection with an assay for detecting respiratory syncytial virus (RSV), wherein a dextran polymeric carrier is used as a “mobile solid phase”, for example, to prepare labeling reagents.